Quantitative Proteomics: Applications and Strategies

1. Quantitative Proteomics: Applications and Strategies Gustavo de SouzaIMM, OUS October 2013

2. A little history… 1985 – First use: up to a 3 kDa peptide could be ionized 1987 – Method to ionize intact proteins (up to 34 kDa) described Instruments have no sequence capability 1989 – ESI is used for biomolecules (peptides) Sequence capability, but low sensitivity 1994 – Term «Proteome» is coined 1995 – LC-MS/MS is implemented «Gold standard» of proteomic analysis

3. 2DE-based approach

4. 2DE-based approach “I see 1000 spots, but identify 50 only.”

5. Column (75 mm)/spray tip (8 mm) Reverse-phase C18 beads, 3 mm No precolumn or split 15 cm Sample Loading:500 nl/min ESI Gradient elution:200 nl/min Platin-wire 2.0 kV LC-MS Fenn et al., Science 246:64-71, 1989.

6. MS-based quantitation Ion Source Mass Analyzer Inlet Detector LC MALDI ES Time-of-Flight Quadrupole Ion Trap Quadrupole-TOF Peak intensities can vary up to 100x between duplicate runs. Quatitative analysis MUST be carried on a single run.

7. Ion Intensity = Ion abundance

8. MS measure m/z Sample 2 Sample 1 Intensity m/z

9. b) a) b) Isotopic Labeling Unlabeled peptide: Labeled peptide: a)

10. Enzymatic Labeling

11. Metabolic Labeling

12. Media with Normal AA () Media with Labelled AA (*) Start SILAC labelling by growing cells in labelling media (labelled AA / dialized serum) m/z m/z * Passage cells to allow incorporation of labelled AA m/z m/z * m/z By 5 cell doublings cells have incorporated m/z * Grow SILAC labelled cells to desired number of cells for experiment m/z m/z SILAC Cells in normal culture media X 3 X 3 Ong SE et al., 2002

13. Chemical Labeling Gygi SP et al., 1999

14. ICAT (Isotope-Coded Affinity Tag)

15. ICAT Thiol-specific group = binds to Cysteins

16. ICAT Thiol-specific group = binds to Cysteins

17. Quantitation at MS1 level Intensity m/z Double sample complexity, i.e. instrument have more “features”to identify, i.e. decrease in identification rate

18. iTRAQ (isobaric Tag for Relative and Absolute Quantitation) RecognizesArg or Lys Total mass of label= 145 Da ALWAYS Sample prep

19. iTRAQ

20. iTRAQ Multiplexing

21. Metabolic VS Chemical Labeling

22. Summary Kolkman A et al., 2005

23. Label-free Mobile phase C18 column, 25cm long A B 20 s A = 5% organic solvent in water B = 95% organic solvent in water Time

24. Label-free Strassberger V et al., 2010

25. Summary

26. Summary

27. Take home message • Quantitation can be done gel-free • Labeling can be performed at protein or peptide level,during normal cell growth or in vitro • Quantitation can be achieved at MS1 or MS2 level • Method choice depends on experimental design, costs, expertise etc • In my PERSONAL OPINION, chemical label should be avoided at all costs unless heavy multiplexing is required

28. State A State B Light Isotope Heavy Isotope Mix 1:1 Optional Protein Fractionation Digest with Trypsin Protein Identification and Quantitation by LC-MS Applications Upregulated protein - Peptide ratio >1 Control vs Tumor Cell? Control vs drug treated cell? Control vs knock-out cell?

29. Applications – Cell Biology Geiger T et al., 2012

30. Applications – Cell Biology

31. Applications – Immunology Meissner et al, Science 2013

32. Clinical Proteomics A. Amyloid tissue stained in Congo Red; B. After LMD. Wisniewski JR et al., 2012

33. Interactomics Schulze and Mann, 2004 Schulze WX et al., 2005

34. Signaling Pathways

35. Take home message • Anything is possible!

36. SILAC Gustavo de SouzaIMM, OUS October 2013

37. Media with Normal AA () Media with Labelled AA (*) Start SILAC labelling by growing cells in labelling media (labelled AA / dialized serum) m/z m/z * Passage cells to allow incorporation of labelled AA m/z m/z * m/z By 5 cell doublings cells have incorporated m/z * Grow SILAC labelled cells to desired number of cells for experiment m/z m/z SILAC Cells in normal culture media X 3 X 3 Ong SE et al., 2002

38. Importance of Dialyzed Serum • non-dialzed serum contains free (unlabeled) amino acids!

39. No alterations to cell phenotype C2C12 myoblast cell line Labeled cells behaved as expected under differentiation protocols

40. Why SILAC is convenient?

41. Why SILAC is convenient? • Convenient - no extra step introduced to experiment, just special medium • Labeling is guaranteed close to 99%. All identified proteins in principle are quantifiable • Quantitation of proteins affected by different stimuli, disruption of genes, etc. • Quantitation of post-translational modifications (phosphorylation, etc.) • Identification and quantitation of interaction partners

42. Catch 22 • SILAC  custom formulation media (without Lys and/or Arg) $$$$$$ • Labeled amino acids – Lys4, Lys6, Lys8, Arg6, Arg10. Use formulation accordingly to media formula (RPMI Lys, 40mg/L) • ***** When doing Arg labeling, attention to Proline conversion! • (50% of tryptic peptides in a random mixture predicted to contain 1 Pro)

43. Proline Conversion!

44. State A State B Light Isotope Heavy Isotope Mix 1:1 Optional Protein Fractionation Digest with Trypsin Protein Identification and Quantitation by LC-MS Typical SILAC experiment workflow Upregulated protein - Peptide ratio >1 Background protein - Peptide ratio 1:1

45. Additional validation criteria * Never use labelled Arg or Lys with same mass difference (Lys6/Arg6)

46. 3 2 Intensity m/z Triple SILAC • Triple Encoding SILAC allows: • Monitoring of three cellular states simultaneously • Study of the dynamics of signal transduction cascades even in short time scales Blagoev B et al., 2004

47. Five time-point “multiplexing” profile Blagoev B et al., 2004

48. Quantitative phosphoproteomics in EGFR signaling 8x 0’ EGF 8x 1’ EGF 0-5-10 min. Cytoplasmic ext. Nuclear extract SCX / TiO2 4x (10 SCX-frac- tions +FT) 8x SCX / TiO2 5’ EGF 8x 5’ EGF 1-5-20 min. Cytoplasmic ext. Nuclear extract SCX / TiO2 44 LC-MS runs SCX / TiO2 8x 10’ EGF 8x 20’ EGF Lysis and Fractionation Anf digestion Phospho- peptide enrichment SILAC- HeLa cells ID and quantitation Blagoev B et al., 2004

49. 40 EGFr-pY1110 ShcA-pY427 ERK1-pY204 ERK2-pY187 EMS1-pS405 10 Relative ratios 2 1 5 10 15 20 EGF (minutes) MAP kinases activation Signal progression

50. Spatial distribution of phosphorylation dynamics Cytosolic STAT5 translocates to the nucleus upon phosphorylation